Receptors are molecules, typically proteins or glycoproteins, found on the surface of cells, including mammalian cells, that possess specific affinity for other molecules known as ligands. Ligands may be small or large (macro) molecules such as proteins. Binding of ligands to receptors on the surface of mammalian cells elicits dramatic responses or "signals" in the cells such as proliferation and adhesion. These cellular responses involve protein-protein interactions and intercellular interactions that regulate important physiological processes, such as the humoral immune response. Defects in receptor structure and function may interfere with recognition of ligands by the cell-bound receptors resulting in disease or dysfunction and death.
Many cell surface receptors and their corresponding ligands have been identified and characterized structurally and biochemically. The study of protein-mediated intercellular signalling yields the surprising result that only short stretches of amino acids, e.g. at least three amino acids, on the surfaces of mammalian proteins are both necessary and sufficient to bind specific receptors and thereby elicit dramatic cellular responses.
Examples of characterized receptors include complement receptor type 2 (CD21 or "CR2") (Weigle et al., In Complement, Muller-Eberhard and Miescher, Eds., Springer-Verlag, Berlin, p. 323 (1985)). The clonal expansion of mature, antigen-reactive B lymphocytes in the humoral immune response is regulated both by direct intercellular interactions (with T helper lymphocytes and accessory cells), and by interactions with soluble growth factors (Unanue, Adv. Immunol., 15:95 (1972)). These soluble factors include B cell growth factors, interleukins, interferons, and components of the complement system (O'Garra et al., Immunol. Today, 8:45 (1988); Weigle et al., supra). Members of the latter two classes of proteins bind specific cell surface receptors, including CR2. CR2 is the B lymphocyte receptor for the proteolytic activation products C3bi, C3dg and C3d of complement component C3 (Cooper et al., Ann. Rev. Immunol., 6:85 (1988); Aggregated C3b and C3d induce B cell proliferation (Erdei et al., Eur. J. Immunol., 15:184 (1985); Melchers et al., Nature, 317:264 (1985)). CR2 is also the receptor for Epstein-Barr virus (EBV), a potent polyclonal B cell activator (Fingeroth et al., Proc. Natl. Acad. Sci. USA, 81:4510 (1985); Frade et al., Proc. Natl. Acad. Sci. USA, 82:1490 (1985)). CR2 plays a central role in signalling B cell proliferation (Cooper et al., supra (1988)). Several monoclonal antibodies and polyclonal anti-CR2 antisera stimulate T cell-dependent B cell proliferation (Cooper et al., supra (1988)). Furthermore, ligand binding to CR2 is necessary for the transition from G.sub.1 to S phase of the B cell cycle of human and murine preactivated blasts (Melchers, supra (1985); Bohnsack and Cooper J. Immunol., 141:2569 (1988)). CR2 is also phosphorylated during B cell stimulation, a common property of growth factor receptors (Changelian and Fearon J. Exp. Med., 161:101 (1986)).
CR2 occurs on normal and malignant B lymphocytes (Cooper et al., supra (1988); Hatzfeld et al , J. Immunol., 140:170 (1988)), on epithelial cells (Young et al., The Lancet, 240 (1986)), and, to a lesser extent, on immature thymocytes and follicular dendritic cells (Tsoukas and Lambris, Eru. J. Immunol., 18:1299 (1988); Reynes et al., J. Immunol., 135:2687 (1985)). The primary structure of CR2 has been deduced from the DNA sequence of its clone. Human CR2 is a membrane glycoprotein of 145 kd, and has sequence similarity to other members of the family of complement binding proteins.
The recognition sites on CR2 for C3d and EBV have been located on the N-terminal part of this longitudinal molecule. The sequence motifs on C3 and EBV coat protein, gp350, that mediate binding to CR2 receptor have also been defined (Lambris et al., Proc. Natl. Acad. Sci. USA, 82:4235 (1985); Nemerow et al., Cell 56:369 (1989)) (Table 1). Synthetic hexapeptides with the sequence of the CR2 binding site on C3 inhibit human and murine B cell proliferation (Lernhardt et al., Immunol. Rev., 99:239 (1987)). Thus CR2 receptor can bind both monomeric C3d and aggregated C3d as ligands, as well as the major epitope of EBV capsid protein.
CR2 ligands act in concert with other B cell growth modulators, including growth factors, lymphokines, and cytokines. Thus, the growth-inducing effect of anti-CR2 monoclonal antibody OKB7 is T cell-dependent, and requires T cell-derived B cell growth factors (Cooper et al., supra (1988)). It has been shown that optimal cell cycle progression and cell division occurs only in the presence of both anti-Ig antibodies and IL-2 or IL-5.
The CR2 receptor is of clinical interest, because it is the receptor for Epstein-Barr virus (EBV) (Frade supra (1985)). EBV is the causative agent of infectious mononucleosis (Huang et al., Int. J. Cancer 14:580 (1974)), and possibly is a human cancer virus, because its presence is correlated with nasopharyngeal carcinoma and Burkitt's lymphoma (Henle et al., Science, 157:1064 (1967)). In addition, EBV may play a role in the onset of B cell neoplasia observed in a substantial fraction of AIDS patients (Yarchoan et al., J. Clin. Invest., 78:439 (1986)). At the least, a substantial fraction of AIDS patients have chronic EBV infections. Exposure of pregnant women to individuals infected with and shedding EBV poses a significant risk to fetal development. It would be useful to better understand the mechanism of CR2 ligand action and to design and engineer proteins that function as recombinant inhibitors of EBV infection and lymphoma proliferation.
Prokaryotic repressors are small, multimeric proteins that are easy to manipulate genetically. Prokaryotic repressors bind short stretches of DNA called operators. Aporepressor proteins bind operators poorly and must complex with other small molecules called corepressors, such as tryptophan or S-adenosylmethionine to form active repressor complexes, or simply, repressors. Corepressors act as "keystones" that fit into and stabilize the hydrophobic cores of their aporepressors.
The E. Coli Tryptophan (Trp) aporepressor monomer is a peptide 108 amino acids long (M.sub.r =12,356 daltons) (Gunsalus and Yanofsky, Proc. Nat. Acad. Sci. USA, 77:7117-7121 (1980)) that assembles as a dimer (Joachimiak et al., Proc. Natl. Acad. Sci. USA, 80:668-672 (1983); Arvidson et al., J. Biol. Chem., 261:238-243 (1986)). Trp aporepressor binds DNA poorly in the absence of the corepressor ligand, L-tryptophan (or the analog 5-methyltryptophan, 5-MT). Aporepressor assembles with tryptophan or 5-MT to form active Trp repressor complex, a global repressor that binds operator sites to regulate the initiation of transcription from at least three different E. coli promoters. In addition, aporepressor can form inactive Trp pseudorepressor complexes with indole-3-propionic acid (IPA) or indole-.pi.-acrylic acid (IAA); these pseudo-repressor complexes bind operator DNA more poorly than aporepressor (Doolittle and Yanofsky, J. Bacteriol., 95:1283-1294 (1968); Baker and Yanofsky, Proc. Natl. Acad. Sci USA, 60:313-320 (1968)).
Trp aporepressor controls three operons that comprise a system to maintain the concentration of L-tryptophan in E. coli homeostatically, within levels necessary for efficient protein synthesis. When concentrations of intracellular tryptophan are low, TrpR exists predominantly as an aporepressor that cannot bind trp operator DNA, and the trpEDCBA biosynthetic genes are expressed maximally. When L-tryptophan levels are high, a substantial fraction of TrpR is active repressor, and tryptophan biosynthesis slows (Cohen and Jacob, C.R. Acad. Sci. Paris, 248:3490-3492 (1959); Yanofsky, J. Amer. Med. Assoc., 218:1026-1035 (1971); Bennet et al., Proc. Natl. Acad. Sci. USA, 73:2351-2355 (1976); Zurawski et al., J. Mol. Biol., 145:47-73 (1981); Yanofsky et al., J. Bacteriol., 158: 1018-1024 (1984)). Trp aporepressor regulates a biosynthetic pathway in response to the amount of an end product; thus, it functions as a rheostat, rather than an on/off switch. In contrast, .mu. and other phage repressors control binary developmental decisions, and are not known to respond to small ligands. Other ligand-activated DNA-binding proteins have been studied to lesser extents.
The X-ray crystal structures of two forms of Trp repressor (Schevitz et al., Nature, 317:782-786 (1985); Lawson et al., Proteins, 3:18-31 (1988)), aporepressor (Zhang et al., Nature, 327:591-597 (1987)), and pseudorepressor (Lawson et al., Nature, 333:869-871 (1988)) have been determined, and show that, when crystallized, the peptide monomer is a bundle of six .alpha.-helices with a disordered, 11-residue N-terminal arm. The TrpR dimer has a remarkable subunit interface, in which four of each subunit's six .alpha.-interface, helices (A, B, C, and F) are interlocked. The amino acid sequence of the two flexible .alpha.-helices, D and E, resembles the conserved "helix-turn-helix" DNA-binding motif characteristic of many prokaryotic repressors, and pairs of the 2.degree. substructures formed by the D loop and E are positioned on the surface of Trp repressor to contact successive major grooves of trp operator DNA. Genetic analyses of mutant TrpR genes show that residues from both D and E are critical for DNA-binding (Bass et al., Science, 242:240-245 (1988); Kelly et al., Proc. Natl. Acad. Sci USA, 79:3120-3124 (1982)).
Recently, Arrowsmith, Jardetsky and coworkers have determined the structure of Trp repressor in solution, using .sup.1 H-NMR spectroscopic methods (Arrowsmith et al., Biochemistry, 29:6332 (1990); Arrowsmith and Jardetsky, submitted for publication (1991). Their results show that the structure of Trp repressor in solution resembles the crystal structures closely, with two major differences. In solution, the first half of A is partially disordered, and the residues organized as D in the crystal do not form an .alpha.-helix, but rather comprise some sort of surface loop (the "D Loop") (Arrowsmith et al., supra (1990)). The binding of the corepressor, L-tryptophan, restricts the motion of the D loop; amide protons of residues in D become less solvent-accessible in the presence of corepressor. However, corepressor binding does not elicit a coil-to-helix transition, because these protons remain uninvolved in H-bond formation in the repressor complex and in the specific repressor/operator complex [Arrowsmith and Jardetsky, supra.
Trp aporepressor is usually stable, and may be purified in large quantities (Arvidson et al., In Protein Purification: Micro to Macro, UCLA Symp. Mol. Cell Biol (Ed. Burgess), Alan R. Liss, NY; (1986); Smith et al., Proc. Natl. Acad. Sci., USA. 82:6104-6108 (1985)). In addition, to understand how particular amino acids contribute to the structure and function of Trp repressor, methods have been developed for both mismatch-primer (Arvidson et al., Genetics, 128 (1991) and cassette-style (Pfau and Youderian, Nuc. Acids Res., 18:6165 (1990) mutagenesis of either single or multiple adjacent codons of TroR. Mutagenesis may be coupled with a rapid screen for Trp repressor function; this screen depends on the color of colonies made by a strain of bacteria, CG103, which overproduces Trp repressor.
Comparisons of the NMR structures of Trp aporepressor and repressor suggests that the binding of indole analogs results in subtle changes in the orientation of D and E relative to the stationary hydrophobic core of the protein. The TrpR dimer has two identical, independent binding sites for corepressor (Arvidson et al., supra (1986); Marmorstein et al., J. Biol. Chem., 262:4922-4927 (1987)); surprisingly, these are formed by the side chains of residues from both monomers in a dimer (Schevitz et al., supra (1985)). The interactions that each corepressor is predicted to make with aporepressor are primarily hydrophobic. Presumably, the binding of corepressor restricts the ensemble of preferred conformations of the DNA-binding domains of an aporepressor to a subset of conformations that bind DNA with lower free energies (pseudorepressor binding restricts aporepressor conformations to a subset that binds DNA with higher free energies).
Attempts have been made to construct hybrids between structural proteins and receptor binding sites. For example, hybrids between proteins having highly repetitive sequences such as silk-like protein (SLP) and the ten-residue RGD motif of fibronectin have been described (Cappello and Crissman, Chemical and Engineering News, pp. 26-32 (July 16, 1990)). Although the hybrid protein is active in vitro, its highly repetitive gene is unstable.
It would be advantageous to provide a method for producing hybrid proteins containing receptor binding sites, that are active as ligands for mammalian cell receptors to design reagents for a variety of applications including treatment of diseases resulting from receptor/ligand dysfunction.